Pharmacological Inhibition Of Akt Research Articles

Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells—either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells.

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

A labdane diterpene exerts ex vivo and in vivo cardioprotection against post-ischemic injury: Involvement of AKT-dependent mechanisms

Therapeutic approaches to protect the heart from ischemia/reperfusion (I/R) injury are an area of intense research, as myocardial infarction is a major cause of mortality and morbidity. Diterpenes are bioactive natural products with great therapeutic potential. In the present study, we have investigated the in vivo cardioprotective effects of a labdane diterpene (DT1) against cardiac I/R injury and the molecular mechanisms involved. DT1 attenuates post-ischemic injury via an AKT-dependent activation of HIF-1α, survival pathways and inhibition of NF-κB signaling. Myocardial infarction (MI) was induced in Wistar rats occluding the left coronary artery (LCA) for 30min followed by 72h reperfusion. DT1 (5mg/kg) was intravenously administered at reperfusion. In addition, we investigated the mechanisms of cardioprotection in the Langendorff-perfused model. Cardioprotection was observed when DT1 was administered after myocardial injury. The molecular mechanisms involved the activation of the survival pathway PDK-1, AKT and AMPK, a reduced phosphorylation of PKD1/2 and sustained HIF-1α activity, leading to increased expression of anti-apoptotic proteins and decreased caspase-3 activation. Pharmacological inhibition of AKT following MI and prior to DT1 challenge significantly decreased the cardioprotection afforded by DT1 therapy at reperfusion. Cardiac function after MI was significantly improved after DT1-treatment, as evidenced by hemodynamic recovery and decreased myocardial infarct size. These findings demonstrate an efficient in vivo cardioprotection by diterpene DT1 against I/R when administered at reperfusion, opening new therapeutic strategies as adjunctive therapy for the pharmacological management of I/R injury.

Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered signaling pathways within tumor cells and their dynamic interaction with the tumor microenvironment. This study evaluated the prognostic value of the phosphorylation status of AKT on Ser473 and Thr308 for the clinical outcome of patients with advanced HNSCC on radiotherapy. Furthermore, we investigated the impact of AKT(Ser473) phosphorylation [p-AKT(Ser473)] in the context of radioresistance using ex vivo tissue cultures that resemble the complex tissue architecture and paracrine interaction with the tumor microenvironment. In a cohort of 120 patients with advanced HNSCC, who were treated with primary or adjuvant radiotherapy, a significant association was found between relative p-AKT(Ser473) levels and overall survival (p = 0.006) as well as progression-free survival (p = 0.021), while no significant correlation was revealed for relative p-AKT(Thr308) levels. In ex vivo tissue cultures p-AKT(Ser473) levels were increased upon irradiation and treatment with the PI3K inhibitor LY294002 inhibited both basal and irradiation induced AKT(Ser473) phosphorylation. Strikingly, pretreatment with LY294002 sensitized tissue cultures derived from primary and recurrent tumors to radiotherapy as determined by impaired tumor cell proliferation and enhanced DNA damage. In conclusion, phosphorylation status of AKT(Ser473) in tumor specimens serves as a novel biomarker to identify patients with advanced HNSCC at high risk for treatment failure following radiotherapy, and our data from ex vivo tissue cultures support the assumption that pharmacological inhibition of AKT(Ser473) phosphorylation might circumvent radioresistance to improve efficiency and reduce toxicity of current treatment modalities.

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

Epithelial‐mesenchymal transition during invasion of cutaneous squamous cell carcinoma is paralleled by AKT activation

Epithelial-mesenchymal transition (EMT) is required for tumour invasion and dissemination to occur. To investigate EMT during invasion of cutaneous squamous cell carcinoma (cSCC) and the involvement of AKT. Using a tissue microarray, we measured expression of EMT-markers and AKT activation in 140 samples from patients with skin cancer and matched samples of normal skin adjacent to cSCC in cSCC in situ (cSCCIS) and in invasive cSCC. We investigated EMT using functional assays and the expression of EMT markers in an isogenic skin cancer progression model using cell lines derived from dysplastic forehead skin (PM1), primary invasive cSCC (MET1) and its lymph node metastasis (MET4). This model was used to investigate AKT-specific inhibition of the EMT process. In comparison with normal skin, and normal skin plus cSCCIS, the invasive cSCCs show significantly increased vimentin expression, decreased E-cadherin expression and increased expression of the active form of AKT. In the cell culture model, the primary MET1 cells display the lowest adhesion potential, the highest migratory and invasive ability through a Matrigel-coated porous membrane, the highest expression of EMT markers vimentin and Slug and the lowest expression of the epithelial marker E-cadherin. Pharmacological AKT inhibition in this model suppressed EMT mechanisms. AKT may serve as a therapeutic target to avoid dissemination of cSCC cells.

Abstract 3328: Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A)

Abstract Background: We have previously shown that the adhesion protein Junctional Adhesion Molecule-A (JAM-A) is over-expressed in a population of aggressive and metastatic breast tumours, and that it regulates the migration of breast cells. JAM-dependent regulation of migration could contribute to the likelihood of metastatic disease, since migration is an early event in metastasis. Our objective was to further interrogate the links between JAM-A signalling and markers of disease progression in breast cancer. Approach and Results: Immunohistochemical analysis of JAM-A in two breast cancer tissue microarrays (TMA) revealed that high JAM-A expression correlated significantly with HER2 protein expression, ERnegativity, high grade breast cancers and aggressive luminal B, HER2 and basal subtypes of breast cancer. Using LCC1 and MCF7-HER2 cells which co-express high levels of JAM-A and HER2, we silenced the gene expression of each one in order to examine potential consequences for protein expression of the other. Knockdown of HER2 had no effect on JAM-A protein levels, but knockdown of JAM-A significantly reduced the protein expression of HER2 and its downstream pro-survival effector pAkt. Accordingly, JAM-A knockdown increased apoptosis via a pathway involving Akt activation, as evidenced by apoptosis normalisation upon pharmacological inhibition of Akt. The expressional regulation of HER2 and pAkt by JAM-A was sensitive to inhibition by the proteasomal inhibitor MG132, suggesting that JAM-A regulates the proteasomal degradation of HER2. Finally, over-expression of wild-type JAM-A was sufficient to significantly enhance proliferative and migratory behaviour in breast cell lines. Conclusions: Our results are consistent with a model whereby JAM-A expression inhibits the proteasomal degradation of HER2, facilitating a pro-survival phenotype through increased Akt activity. Therefore JAM-A antagonists may have future therapeutic value in the treatment of breast tumours overexpressing HER2. Citation Format: Kieran Brennan, Elaine A. McSherry, Lance Hudson, Elaine W. Kay, Leonie S. Young, Arnold DK Hill, Ann M. Hopkins. Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2014-3328

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation.

Zinc Modulates High Glucose-Induced Apoptosis by Suppressing Oxidative Stress in Renal Tubular Epithelial Cells

Hyperglycemia is a characteristic of diabetic nephropathy, inducing renal tubular cell apoptosis by eliciting oxidative stress and inflammation. Zinc (Zn) is known as an essential trace element in many enzymes and proteins involved in antioxidant defenses, electron transport, and exerting antiapoptotic or cytoprotective effects. In this study, the underlying mechanisms involved in the protective effects of Zn on high glucose-induced cytotoxicity were explored using cultured renal tubular epithelial cells (NRK-52E). The authors discovered that Zn supplementation inhibited high glucose (HG)-induced NRK-52E cell apoptosis by attenuating reactive oxygen species production, inhibiting HG-induced caspase-3 and caspase-9 activation, and inhibiting the release of cytochrome c from mitochondria to the cytosol. Further analysis revealed that Zn supplementation facilitated cell survival through increasing nuclear translocation of NF-E2-related factor 2 (Nrf2), leading to increased regulation of levels of two antioxidant enzymes, hemeoxygenase-1 and glutamate cysteine ligase, which provided an adaptive survival response against the HG-induced oxidative cytotoxicity. Moreover, the Zn-mediated increases in Nrf2 activity were suppressed by the pharmacological inhibition of Akt or extracellular signal-regulated kinase 1/2. Taken together, these findings suggest that Zn antiapoptosis capacity through the activation of Akt and ERK signal pathways leads to Nrf2 activation and, subsequently, Nrf2 target gene induction, thereby protecting the NRK-52E cells from HG-induced apoptosis.

Thymoquinone induces heme oxygenase-1 expression in HaCaT cells via Nrf2/ARE activation: Akt and AMPKα as upstream targets

Thymoquinone (TQ), an active constituent of Nigella sativa, possesses anti-inflammatory and anticancer properties. Multiple lines of evidence suggest that the induction of heme oxygenase-1 (HO-1) suppresses inflammation and carcinogenesis. In the present study, we examined the effect of TQ on HO-1 expression in human keratinocytes (HaCaT) and elucidated its underlying molecular mechanisms. TQ induced the expression of HO-1 in HaCaT cells in a concentration- and time-dependent manner. Treatment with TQ increased the localization of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) in the nucleus and elevated the antioxidant response element (ARE)-reporter gene activity. Knockdown of Nrf2 abrogated TQ-induced HO-1 expression and the ARE luciferase activity. TQ induced the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and cyclic AMP-activated protein kinase-α (AMPKα). Pharmacological inhibition of Akt or AMPKα, but not that of ERK, abrogated TQ-induced nuclear localization of Nrf2, the ARE-luciferase activity and the expression of HO-1. TQ also generated reactive oxygen species (ROS) and pretreatment with N-acetyl cysteine (NAC) abrogated TQ-induced ROS accumulation, Akt and AMPKα activation, Nrf2 nuclear localization, the ARE-luciferase activity, and HO-1 expression in HaCaT cells. Taken together, TQ induces HO-1 expression in HaCaT cells by activating Nrf2 through ROS-mediated phosphorylation of Akt and AMPKα.

AKT activation promotes PTEN hamartoma tumor syndrome–associated cataract development

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.

PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere

IntroductionActivation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo.Case preparationTo further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere.ConclusionOur results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

Neuronal ablation of p-Akt at Ser473 leads to altered 5-HT1A/2A receptor function

The serotonergic system regulates a wide range of behavior, including mood and impulsivity, and its dysregulation has been associated with mood disorders, autism spectrum disorder, and addiction. Diabetes is a risk factor for these conditions. Insulin resistance in the brain is specifically associated with susceptibility to psychostimulant abuse. Here, we examined whether phosphorylation of Akt, a key regulator of the insulin signaling pathway, controls serotonin (5-HT) signaling. To explore how impairment in Akt function regulates 5-HT homeostasis, we used a brain-specific rictor knockout (KO) mouse model of impaired neuronal phosphorylation of Akt at Ser473. Cortical 5-HT1A and 5-HT2A receptor binding was significantly elevated in rictor KO mice. Concomitant with this elevated receptor expression, the 5-HT1A receptor agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) led to an increased hypothermic response in rictor KO mice. The increased cortical 5-HT1A receptor density was associated with higher 5-HT1A receptor levels on the cortical cell surface. In contrast, rictor KO mice displayed significantly reduced head-twitch response (HTR) to the 5-HT2A/C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), with evidence of impaired 5-HT2A/C receptor signaling. In vitro, pharmacological inhibition of Akt significantly increased 5-HT1A receptor expression and attenuated DOI-induced 5-HT2A receptor signaling, thereby lending credence to the observed in vivo cross-talk between neuronal Akt signaling and 5-HT receptor regulation. These data reveal that defective central Akt function alters 5-HT signaling as well as 5-HT-associated behaviors, demonstrating a novel role for Akt in maintaining neuronal 5-HT receptor function.

Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

HIF‐2α regulates hypoxia‐induced arginase II expression through EGFR

To test the hypothesis that arginase II is induced in an oxygen‐dependent manner in human pulmonary microvascular endothelial cells (hPMVEC), cells were incubated in either normoxia (20% O2) or hypoxia (1% O2) for 6 or 24 h, and some of the hypoxic hPMVEC were returned to normoxia or hypoxia for an additional 24 h. The hPMVEC incubated in hypoxia for 24 h produced ~70% less NO than cells incubated in normoxia (p<0.01). Greater HIF‐2α, EGFR, and arginase II protein levels were found in hypoxic hPMVEC than in normoxic cells (p<0.05). Hypoxic hPMVEC returned to normoxia had substantially greater NO production, while arginase II, HIF‐2α, and EGFR protein levels were lower, than in hypoxic hPMVEC (p<0.05). hPMVECs were transfected with siRNA for 48 h to knockdown the expression of HIF‐2α or EGFR. We found that silencing HIF‐2α prevented hypoxia‐induced EGFR and arginase II expression. Conversely, silencing of EGFR also prevented HIF‐2α and arginase II expression, as well as downstream ERK and Akt activation. These results suggest that there is a feedback regulation loop between HIF‐2α and EGFR. Pharmacological inhibition of Akt and MEK1/2 prevented hypoxia‐induced arginase II expression. Thus, arginase II is induced by hypoxia through a pathway mediated by HIF2α, EGFR, ERK, and Akt. We speculate that arginase II regulation may represent a potential therapy for pulmonary hypertension associated with hypoxia.

VAMP-Associated Protein B (VAPB) Promotes Breast Tumor Growth by Modulation of Akt Activity

VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer.

Inhibition of Mycobacterial Infection by the Tumor Suppressor PTEN

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.